JP3127238B2 - Methods and detectors for separation processes - Google Patents

Abstract

PCT No. PCT/SE93/00305 Sec. 371 Date Oct. 7, 1994 Sec. 102(e) Date Oct. 7, 1994 PCT Filed Apr. 7, 1993 PCT Pub. No. WO93/20435 PCT Pub. Date Oct. 14, 1993The present invention comprises a method Of detecting and quantifying components separated by capillary electrophoresis or other techniques using thin capillaries in the separation. The method of the present invention comprises irradiating the whole or large parts of the capillary at short time intervals and detecting the fluorescence originating from the sample components, preferably by a CCD detector (charge coupled device). Thereby, an instantaneous image of the progress of the separation process is obtained. The present invention further comprises an apparatus for performing this detection.

Description

DETAILED DESCRIPTION OF THE INVENTION In the field of biological science, there is a considerable need to analyze complex mixtures of biomolecules. Examples are the analysis of metabolites, proteins, antibodies and drugs in clinical analysis. Another example is the analysis of protein composition in fermentation of proteins identified by genetic engineering.

A common method for performing bioanalysis is to separate a sample into its components and then quantify the components. Conventional separation methods are chromatography and electrophoresis. Electrophoresis is a powerful analytical method. The reason is that the separation power of the method is high, which means in particular that many components can be analyzed at once and in the same test. The method can also be very sensitive when combined with a detection method suitable for the separated material. On the other hand, the method is generally slow, with typical separation times of 1 to 10 hours.

During the last years, a particular form of electrophoresis, capillary electrophoresis, has received considerable attention. The electrophoresis process occurs in a thin capillary of quartz (inner diameter: about 0.005-0.1 mm). The thin capillaries easily cool down the Joule heat generated during the electrophoresis process. Thus, very high electric field strengths, for example 300 V / cm, can be used in electrophoretic separations. High electric field strengths give very rapid migration velocities, so that capillary electrophoresis advantageously reduces the separation time. In normal electrophoresis, temperature gradients readily occur in the separation layer. Temperature gradients significantly reduce the separation of sample components.
This problem is solved by capillary electrophoresis, where the thin capillaries cool easily and thus do not cause the temperature gradients in question. As a result, the separation capacity is also very good in capillary electrophoresis. The real papers are H. Carchon and
Written by E. Eggermont [International Chromium
atography Laboratory, Vol. 6, pp. 17-22 (1991
Year)〕.

Detection and quantification of mixtures separated by capillary electrophoresis are usually carried out using an ultraviolet / visible detector or a fluorescent detector. 8, No. 10, pages 788-799 (1990), DMGoodall, DKLloyd and SJW
illiams]. These detectors are always located at one end of the capillary. Normally, the light beam passes vertically through the capillary and the detector measures the amount of light passed or the amount of fluorescent light emitted. Thereby, the separated substances are sequentially detected as they pass through the detector.
This means that electrophoresis must continue until all physics has passed the detector. Often the number of substances in the sample is unknown, so that the electrophoresis test must be performed much longer than is actually needed to ensure that all sample components pass through the detector. Thus, it is shown that the limiting factor of the capillary electrophoresis, that is, the principle of continuous detection, in other words, the sample components are sequentially detected. Alternatively, if a detection principle is obtained in which the entire capillary is examined instantaneously by a particular detector, a similar detection principle is obtained, in which all components are detected simultaneously. The present invention discloses such principles and the construction of suitable devices.

The novel detection principle consists in examining the entire capillary at short intervals and monitoring capillary electrophoresis with a detector that records the location of the sample components in the capillary. At the same time, the detector records the spectral characteristics of the sample components, which facilitates their identification. The obtained data is stored in a computer.

To illustrate the features of the function, the following short device description is considered as guidance (further detailed description will be given): the detector of the invention consists of a strong light source, for example a laser. The laser preferably irradiates the entire capillary at short time intervals. With each irradiation, the spatially separated sample material emits fluorescence. The fluorescence emission from the capillary is focused through a lens system onto a CCD (charge coupled device) detector, forming an electronic image of the capillary appearance. The computer stores this image. During each irradiation time, the sample components move slightly and this movement is recorded by the CCD detector. The computer continuously converts the stored data and can show the progress of the separation in real time on the screen of the display. This is a very great advantage. If the separation of the sample components is good enough to quantify them with sufficient precision, it can be seen directly on the computer screen. In some cases, the computer may make this determination on its own. This means a great saving of time since the separation can be stopped much earlier than with a detector located at the end of the capillary. This is especially true if the sample contains components that migrate very slowly.

In the present invention, the polarity of the electrophoresis process can be reversed when a sufficiently good separation has been achieved, and the sample can be flushed back to prepare the capillary for the next sample. It is easily understood that the present invention improves efficiency (sample / hour) by as much as 10-fold when the sample contains slowly migrating substances.

Another advantage of the novel detector is that the olerepeater / computer has excellent control of the separation process. The problem with all separation processes involving continuous detection, such as conventional capillary electrophoresis, is that it is not possible to confirm that all components are actually reaching the detector when the separation is stopped. This problem is effectively solved by monitoring the entire separation capillary according to the invention.

There are several areas in which the new detectors can be used in particular. One of them is sequence analysis of genes, where analysis speed in combination with reliable assays is very important. Controlled interruption
ed replication) Samples generated by the Sanger method consist of a mixture of oligonucleotides of various lengths. Oligonucleotides are fluorescent dyes, such as argon lasers (51
It is labeled with rhodamine 110, rhodamine 6G, tetramethylrhodamine and X-rhodamine which are well adapted to excitation by 4 nm). Very fast separation cycles are possible here. As soon as sufficient separation is achieved, the system can be regenerated by reversing the electrodes. As a result, sequences that are considerably longer than those that are usually processed practically can be handled.

Another very interesting area is the study of protein-ligand interactions. If the protein has a higher migration rate than the ligand, the ligand is first added to the capillary. After a while, the protein is added. The protein migrates into the slowly migrating ligand segment,
After a while it passes completely through the ligand segment. Associated with its passage, interactions occur that reflect the migration rate of the components. The new detector can track these interactions very accurately. Then, by analyzing the resulting fronts and their composition, very interesting information can be extracted regarding the equilibrium and kinetic rate constants.

The above description of the invention relates to capillary electrophoresis. The advantage of the same origin applies to electrokinetic chromatography, a separation method using a capillary electrophoresis device, where the actual separation process is by chromatography rather than electrophoresis. Separation by HPLC techniques using thin glass or silica columns can also be used as a novel detection method.

There are many other aspects of the invention, including various ways of connecting a light source (laser or other light source) to the capillary. One way is to irradiate the fiber perpendicular to its longitudinal direction. Another method is to introduce an excitation method to the end face of the capillary. Since this method minimizes the amount of stray light, it is expected that particularly uniform and effective irradiation will be performed simultaneously.
Many types of lasers can be used as the excitation source, the choice of which is determined inter alia by the desired excitation wavelength. HeCd lasers (eg, from Liconix) have 442 and
Give the appropriate line at 325 nm. A small argon ion laser (eg, from Spectra Physics) gives a blue-green line, but ultraviolet light is also obtained around 350 nm. A pulsating nitrogen laser (eg, Laser Science) emits at 337 nm,
It can also be used to pump dye lasers that generate any visible wavelength.

Without discrimination of the wavelength, all of the fluorescence is imaged on a linear detector (diode array) after the excitation laser light is suppressed by a sharp broad-pass filter, preferably a color filter (eg from Schott). Made. Diode strings suitable for this purpose are sold, for example, by EG & G or Reticon. Diode arrays are usually 25nm long
It has 1024 diodes, but longer designs are also available. The lens system has straight capillaries,
Each diode is selected to create an image on the diode array with the correct reduction so that it corresponds to a particular location along the capillary. The spatially separated detector signals are read by a computer via a control unit. If a pulsating excitation source (eg, a nitrogen laser) is used, a gated microchannel plate can be used in front of the diode rows to effectively amplify the fluorescence while simultaneously suppressing background light.

Detector systems are particularly useful where the color distribution of the fluorescent spectrum can also be achieved, so that spatially separated signals are recorded at the same time. This can be done by creating an image of the capillary on the entrance slit of the spectrometer. This slit is arranged in parallel to a grating groove for spectrally decomposing light perpendicular to the slit direction. At the focal plane, the light falls on a two-dimensional CCD detector in one dimension corresponding to various locations along the capillary, and the corresponding spectrum in the other dimension. The spectra at various positions are read via the reading unit. A spectrometer (imaging spectrometer) arranged in this way is known from a remote analysis sensor mounted on a satellite. The computer evaluates the spectra in a rigorous analysis and compares them to a stored reference spectrum. Alternatively, the intensity in a predetermined wavelength range is read corresponding to the emission band of a fluorescent label having a known emission.

Limited spectral analysis is also achieved by utilizing a linear detector (diode array) where the fluorescence is first split by a particular mirror system, for example into four parts, each focused on an image line. Can be done. Due to the precise positioning of the mirrors, these image lines can be arranged in sequence along a linear detector. In four separate beams, a bandpass filter is introduced, which separates the fluorescence from the fluorescence labels used, for example, for DNA sequence analysis. In this way,
A spatially separated image is obtained at the same time, from which the positions of the various labels along the capillary are known. Here, the beam splitting and the recording method of the fluorescent light are performed by the technique (fluorescent imaging system) disclosed in Swedish Patent No. 455,646.

As described above, when the proper separation of each component has been achieved, the signal is read and analyzed. However, because each component migrates at a uniform rate, the capillary information is read several times over a long time interval and is calculated together by a computer into a signal indicating the preferred combination of signal strength and resolution. In this way, optimal functions with respect to speed and resolution can be achieved in a measuring instrument according to the principles of the present invention.

FIG. 1 shows an experimental configuration using a diode array as a detector. A laser light source (1) associated with an optical element illuminates a capillary electrophoresis capillary (2). Thereafter, the substance (3) present in the capillary emits fluorescence. The fluorescent light is focused by a lens system (4) on a diode array (5). The signal from the diode array is sent to a computer (6) which can indicate in real time the location of the separated substance (3) on the screen of the display.

FIG. 2 shows an experimental configuration using a cooled CCD camera as a detector. The power unit (1) applies a voltage to the capillary (2) in which the separation of the sample components takes place. The laser light source (3) passes through the modulation lens system (4) and the capillary (2)
Is irradiated. Fluorescence from the sample components that fluoresce in the capillary (2) passes through the lens system (5) and the optical amplifier (6) and enters the CCD camera (7) using a cooled detector unit. The signal from the CCD camera is sent to a computer (8) equipped with an image processing program. The display screen of the computer shows the separation process in the capillary (2) in real time.

Of course, the present invention is not limited to the embodiments described and shown in the drawings, but many changes and modifications may be made without departing from the general concept of the invention as defined in the claims. .

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Larson, Per-Ulov Sweden S-226 53 Lund. Vogelfunds-Vueien 56 (72) Inventor Miyabayashi, Akiyoshi Sweden S-217 51 Malmo. (72) Inventor Moosbach, Klaus S. Sweden 244 94 Fürlund. Ratsuka Brick 31-38 (72) Inventor Nilsson, Statuean Sweden 224-57 Lund. Siriusgatan 14 (72) Inventor Svemberberg, Sune Sweden-222 25 Lund. -. Gustav Sweden countries es -224 71 Lund Son Galle Vu forever 6 er (58) investigated the field (Int.Cl. ^7, DB name) G01N 27/447 G01N 21/17 G01N 21/64 G01N 30/74

Claims (12)

(57) [Claims] 1. A method for separating sample components in a thin capillary by irradiating the capillary with, for example, capillary electrophoresis, electrokinetic capillary chromatography or capillary column chromatography.
A method of analyzing a sample by detecting light emitted or absorbed by the sample components, wherein the light is simultaneously and quickly removed during the separation process for all or at least a majority of the length of the separation capillary. A method characterized in that it detects and forms a separation pattern and thus a continuous instantaneous image of the progress of the separation, whereby the separation can be stopped when the desired separation of the sample components has been achieved. 2. The method according to claim 1, wherein the detection is based on the fluorescence of the sample components. 3. The method according to claim 1, wherein the detection is based on light scattering of the sample components. 4. The method according to claim 1, wherein the irradiation is performed by a laser.
The method according to any one of claims 1 to 3. 5. The method according to claim 1, wherein the principle of separation is capillary electrophoresis.
The method according to any one of claims 4 to 4. 6. The method according to claim 1, wherein the separation principle is capillary column chromatography. 7. Separation capillaries, especially for capillary electrophoresis, electrokinetic chromatography or capillary column chromatography, light sources for irradiating capillaries, and light emitted or absorbed by sample components. A detector for detecting, wherein the light source illuminates the entire separation capillary or at least a majority thereof, and the detector simultaneously simultaneously separates the entire separation capillary or at least a majority thereof.
A sample analyzer characterized by being an imaging detector that records light emitted or absorbed by a sample component in a short time to form a separation pattern and thus a continuous instantaneous image of the progress of separation. . 8. The apparatus according to claim 7, wherein the imaging detector is a two-dimensional CCD (charge coupled device). 9. A CCD detector wherein an image of a separation capillary having a sample component spatially separated on its entrance slit is created for spectral separation perpendicular to the slit of light emitted from the sample component; 9. The apparatus according to claim 8, wherein a spectrometer is preceded by which spatial separation is achieved in one direction of the two-dimensional detector and spectral separation is achieved in the other direction. 10. The apparatus according to claim 7, wherein the imaging detector is a diode array. 11. A diode array (i) optically splits light emitted from a sample component into at least two, preferably four, parts, each of which is arranged in turn in a diode row. 11. The apparatus according to claim 10, wherein a beam splitting system adapted to focus on the image line and (ii) filtering means for spectrally filtering each of said portions of emitted light are each preceded. 12. A computer unit for processing recorded data from an imaging detector together for characterization of each sample component based on its spectral characteristics and classification by comparison with spectral information stored in a computer. An apparatus according to any one of claims 7 to 11, comprising: